Inhibitory Effects, Fluorescence Studies, and Molecular Docking Analysis of Some Novel Pyridine-Based Compounds on Mushroom Tyrosinase.

Melanin biosynthesis in different organisms is performed by a tyrosinase action. Excessive enzyme activity and pigment accumulation result in different diseases and disorders including skin cancers, blemishes, and darkening. In fruits and vegetables, it causes unwanted browning of these products and reduces their appearance quality and economic value. Inhibiting enzyme activity and finding novel powerful and safe inhibitors are highly important in agriculture, food, medical, and pharmaceutical industries. In this regard, in the present study, some novel synthetic pyridine-based compounds including 2,6-bis (tosyloxymethyl) pyridine (compound 3), 2,6-bis (butylthiomethyl) pyridine (compound 4), and 2,6-bis (phenylthiomethyl) pyridine (compound 5) were synthesized for the first time, and their inhibitory potencies were assessed on mushroom tyrosinase diphenolase activity. The results showed that while all tested compounds significantly decreased the enzyme activity, compounds 4 and 5 had the highest inhibitory effects (respectively, 80 and 89% inhibition with the IC50 values of 17.0 and 9.0 µmol L-1), and the inhibition mechanism was mixed-type for both compounds. Ligand-binding studies were carried out by fluorescence quenching and molecular docking methods to investigate the enzyme-compound interactions. Fluorescence quenching results revealed that the compounds can form nonfluorescent complexes with the enzyme and result in quenching of its intrinsic emission by the static process. Molecular docking analyses predicted the binding positions and the amino acid residues involved in the interactions. These compounds appear to be suitable candidates for more studies on tyrosinase inhibition.

Tyrosinase-Based Proximity Labeling in Living Cells and In Vivo.

Characterizing the protein constituents of a specific organelle and protein neighbors of a protein of interest (POI) is essential for understanding the function and state of the organelle and protein networks associated with the POI. Proximity labeling (PL) has emerged as a promising technology for specific and efficient spatial proteomics. Nevertheless, most enzymes adopted for PL still have limitations: APEX requires cytotoxic H2O2 for activation and thus is poor in biocompatibility for in vivo application, BioID shows insufficient labeling kinetics, and TurboID suffers from high background biotinylation. Here, we introduce a bacterial tyrosinase (BmTyr) as a new PL enzyme suitable for H2O2-free, fast (≤10 min in living cells), and low-background protein tagging. BmTyr is genetically encodable and enables subcellular-resolved PL and proteomics in living cells. We further designed a strategy of ligand-tethered BmTyr for in vivo PL, which unveiled the surrounding proteome of a neurotransmitter receptor (Grm1 and Drd2) in its resident synapse in a live mouse brain. Overall, BmTyr is one promising enzyme that can improve and expand PL-based applications and discoveries.

Integrating a Copper-Histidine Brace in a Mimetic Nanozyme Streamlines the Tyrosinase Recognition Moiety to Achieve Chiral Differentiation.

Designing artificial mimetic enzymes with high activity/selectivity to replace chiral bioenzymes is of great interest in the development of chiral materials consisting of molecules, enantiomers, that exist in two forms as mirror images of one another but cannot be superimposed. In this study, the chiral catalytic structural unit was streamlined from tyrosinase to integrate a mimetic nanozyme. The chiral amino acid l-histidine, as the chiral binding/recognition site, and the active metal site Cu were coupled (Cu@l-His) to create a copper-histidine brace with enantioselective catalytic ability to tyrosinol enantiomers. Results of kinetic parameters and activation energies confirmed the excellent peroxidase-like activity with a preference of Cu@l-His to l-tyrosinol. Such a preference could be attributed to the structurally oriented copper-histidine brace with a stronger affinity and catalytic activity to l-tyrosinol. By accurately evaluating chiral recognition units derived from bioenzymes, stable and superior chiral mimetic nanoenzymes could be constructed in a more straightforward and simplified manner, and they could also be extended to the reconstruction of diverse chiral enzymes.

Tyrosinase regulates the motility of human melanoma cell line A375 through its hydroxylase enzymatic activity.

Melanoma, originating from melanocytes, is a highly aggressive tumor. Tyrosinase is involved in melanin production in melanocytes, and its overexpression is noted in malignant melanomas. However, the role of tyrosinase in melanomas remains unclear. Therefore, this study aimed to evaluate the potential functions of tyrosinase in the human melanoma cell line A375. The expression level of tyrosinase in A375 cells was undetectable. However, markedly increased expression level was observed in the mouse melanoma cell line B16F10 and the human melanoma cell line WM266-4. Subsequently, we investigated the effect of ectopic tyrosinase expression on A375 cell motility using wound-healing assay. The overexpression of tyrosinase resulted in enhanced cell migration in both stable and transient tyrosinase expression cells. The levels of filamentous actin were decreased in tyrosinase-expressing A375 cells, suggesting that tyrosinase regulates cell motility by modulating actin polymerization. Histidine residues in tyrosinase are important for its enzymatic activity for synthesizing melanin. Substitution of these histidine residues to alanine residues mitigated the promotion of tyrosinase-induced A375 cell metastasis. Furthermore, melanin treatment enhanced A375 cell metastasis and phosphorylation of Cofilin. Thus, our findings suggest that tyrosinase increases the migration of A375 cells by regulating actin polymerization through its enzymatic activity.

Mushroom Tyrosinase: Six Isoenzymes Catalyzing Distinct Reactions.

"Mushroom tyrosinase" from the common button mushroom is the most frequently used source of tyrosinase activity, both for basic and applied research. Here, the complete tyrosinase family from Agaricus bisporus var. bisporus (abPPO1-6) was cloned from mRNA and expressed heterologously using a single protocol. All six isoenzymes accept a wide range of phenolic and catecholic substrates, but display pronounced differences in their specificity and enzymatic reaction rate. AbPPO3 ignores γ-l-glutaminyl-4-hydroxybenzene (GHB), a natural phenol present in mM concentrations in A. bisporus, while AbPPO4 processes 100 µM GHB at 4-times the rate of the catechol l-DOPA. All six AbPPOs are biochemically distinct enzymes fit for different roles in the fungal life cycle, which challenges the traditional concept of isoenzymes as catalyzing the same physiological reaction and varying only in secondary properties. Transferring this approach to other enzymes and organisms will greatly stimulate both the study of the in vivo function(s) of enzymes and the application of these highly efficient catalysts.

Interactions of Phenylalanine Derivatives with Human Tyrosinase: Lessons from Experimental and Theoretical tudies.

The pigmentation of the skin, modulated by different actors in melanogenesis, is mainly due to the melanins (protective pigments). In humans, these pigments' precursors are synthetized by an enzyme known as tyrosinase (TyH). The regulation of the enzyme activity by specific modulators (inhibitors or activators) can offer a means to fight hypo- and hyper-pigmentations responsible for medical, psychological and societal handicaps. Herein, we report the investigation of phenylalanine derivatives as TyH modulators. Interacting with the binuclear copper active site of the enzyme, phenylalanine derivatives combine effects induced by combination with known resorcinol inhibitors and natural substrate/intermediate (amino acid part). Computational studies including docking, molecular dynamics and free energy calculations combined with biological activity assays on isolated TyH and in human melanoma MNT-1 cells, and X-ray crystallography analyses with the TyH analogue Tyrp1, provide conclusive evidence of the interactions of phenylalanine derivatives with human tyrosinase. In particular, our findings indicate that an analogue of L-DOPA, namely (S)-3-amino-tyrosine, stands out as an amino phenol derivative with inhibitory properties against TyH.

Probiotics from kefir: Evaluating their immunostimulant and antioxidant potential in the carpet shell clam (Ruditapesdecussatus).

This study aimed to investigate the impact of incorporating kefir into the diet on biometric parameters, as well as the immune and antioxidant responses of the carpet shell clam (Ruditapes decussatus) after an experimental infection by Vibrio alginolyticus. Clams were divided into a control group and a treated group. The control group was fed on spirulina (Arthrospira platensis) alone. While, the treated group was fed on spirulina supplemented with 10% dried kefir. After 21 days, clams were immersed in a suspension of V. alginolyticus 5 × 105 CFU mL -1 for 30 min. Seven days after experimental infection, survival was 100% in both groups. The obtained results showed a slight increase in weight and condition index in clams fed with kefir-supplemented diet for 21 days compared to control clams. Regarding antioxidant responses, the treated group showed higher superoxide dismutase activity compared to the control group. However, the malondialdehyde level was lower in the treated clams than in the control. In terms of immune parameters, the treated group showed slightly elevated activities of phenoloxidase, lysozyme and alkaline phosphatase, whereas a decreased lectin activity was observed compared to the control group. The obtained results suggest that kefir enhanced both the antioxidant and immune response of infected clams.

Fast freezing inhibits melanin synthesis of melanocytes by modulating the Wnt/ß-catenin signalling pathway.

Skin hyperpigmentation is mainly caused by excessive synthesis of melanin; however, there is still no safe and effective therapy for its removal. Here, we found that the dermal freezer was able to improve UVB-induced hyperpigmentation of guinea pigs without causing obvious epidermal damage. We also mimic freezing stimulation at the cellular level by rapid freezing and observed that freezing treatments <2.5 min could not decrease cell viability or induce cell apoptosis in B16F10 and Melan-A cells. Critically, melanin content and tyrosinase activity in two cells were greatly reduced after freezing treatments. The dramatic decrease in tyrosinase activity was associated with the downregulation of MITF, TYR, TRP-1 and TRP-2 protein expression in response to freezing treatments for two cells. Furthermore, our results first demonstrated that freezing treatments significantly reduced the levels of p-GSK3ß and ß-catenin and the nuclear accumulation of ß-catenin in B16F10 and Melan-A cells. Together, these data suggest that fast freezing treatments can inhibit melanogenesis-related gene expression in melanocytes by regulating the Wnt/ß-catenin signalling pathway. The inhibition of melanin production eventually contributed to the improvement in skin hyperpigmentation induced by UVB. Therefore, fast freezing treatments may be a new alternative of skin whitening in the clinic in the future.

TYR mutation in a Chinese population with oculocutaneous albinism: Molecular characteristics and ophthalmic manifestations.

Oculocutaneous albinism (OCA) is a rare inherited disorder characterized by a partial or complete reduction of melanin biosynthesis that leads to hypopigmentation in the skin, hair and eyes. The OCA1 subtype is caused by mutations in TYR. The purpose of this study was to investigate the genetic and clinical ophthalmic characteristics of TYR mutations in patients with OCA. Herein, 51 probands with a clinical diagnosis of OCA were enrolled. Whole-exome sequencing and comprehensive ophthalmic examinations were performed. Overall, TYR mutations were detected in 37.3% (19/51) in the patients with OCA. Fifteen patients had compound heterozygous variants, and four cases had homozygous variants. Eleven different pathogenic variants in TYR were detected in these 19 patients, with missense, insertion, delins and nonsense in 71.1% (27/38), 15.8% (6/38), 2.6% (1/38), and 10.5% (4/38), respectively. Clinical examinations revealed that 84.2% (16/19) of patients were OCA1A, and 15.8% (3/19) were OCA1B. Most TYR probands (52.6%, 10/19) had moderate vision impairment, 15.8% (3/19) had severe visual impairment, 10.5% (2/19) exhibited blindness, only 5.3% (1/19) had mild visual impairment and 15.8% (3/19) were not available. Photophobia and nystagmus were found in 100% (19/19) of the patients. In addition, grade 4 foveal hypoplasia was detected in 100% (12/12) of the patients. In conclusion: The TYR patients exhibited severe ocular phenotypes: the majority (93.8%, 15/16) of them had a moderate vision impairment or worse, and 100% (12/12) had severe grade 4 foveal hypoplasia. These novel findings could provide insight into the understanding of OCA.

Disposable biosensor based on ionic liquid, carbon nanofiber and poly(glutamic acid) for tyramine determination.

In this study, an electrochemical biosensor based on carbon nanofibers (CNF), ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate (IL), poly(glutamic acid) (PGA) and tyrosinase (Tyr) modified screen printed carbon electrode (SPE) was constructed for tyramine determination. Optimum experimental parameters such as CNF and IL amount, polymerization conditions of glutamic acid, enzyme loading, pH of test solution and operating potential were explored. The construction steps of the Tyr/PGA/CNF-IL/SPE were pursued by scanning electron microscopy and cyclic voltammetry. The Tyr/PGA/CNF-IL/SPE biosensor exhibited linear response to tyramine in the range of 2.0 × 10-7 - 4.8 × 10-5 M with a low detection limit of 9.1 × 10-8 M and sensitivity of 302.6 µA mM-1. The other advantages of Tyr/PGA/CNF-IL/SPE include its high reproducibility, good stability and anti-interference ability. The presented biosensor was also applied for tyramine determination in malt drink and pickle juice samples and mean analytical recoveries of spiked tyramine were calculated as 100.6% and 100.4% respectively.

Disposable biosensor based on nanodiamond particles, ionic liquid and poly-l-lysine for determination of phenolic compounds.

This study describes the development of a highly sensitive amperometric biosensor for the analysis of phenolic compounds such as catechol. The biosensor architecture is based on the immobilization of tyrosinase (Tyr) on a screen-printed carbon electrode (SPE) modified with nanodiamond particles (ND), 1-butyl-3-methylimidazolium hexafluorophosphate (IL) and poly-l-lysine (PLL). Surface morphologies of the electrodes during the modification process were evaluated by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to investigate the electrochemical characteristics of the modified electrodes. Owing to the synergistic effect of the modification materials, the Tyr/PLL/ND-IL/SPE exhibited high sensitivity (328.2 µA mM-1) towards catechol with a wide linear range (5.0 × 10-8 - 1.2 × 10-5 M) and low detection limit (1.1 × 10-8 M). Furthermore, the method demonstrated good reproducibility and stability. The amperometric response of the biosensor towards other phenolic compounds such as bisphenol A, phenol, p-nitrophenol, m-cresol, p-cresol and o-cresol was also investigated. The analytical applicability of the biosensor was tested by the analysis of catechol in tap water. The results of the tap water analysis showed that the Tyr/PLL/ND-IL/SPE can be used as a practical and effective method for catechol determination.

(2-Methylbutyryl)shikonin Naturally Occurring Shikonin Derivative Ameliorates the α-MSH-Induced Melanogenesis via ERK1/2 and p38 MAP Kinase-Mediated Down-Regulation of the MITF Transcription Factor.

Cutaneous pigmentation is an important phenotypic trait whose regulation, despite recent advances, has yet to be completely elucidated. Melanogenesis, a physiological process of melanin production, is imperative for organism survival as it provides protection against the environmental insults that majorly involve sunlight-induced skin photodamage. However, immoderate melanin synthesis can cause pigmentation disorders associated with a psychosocial impact. In this study, the hypopigmentation effect of (2-methylbutyryl)shikonin, a natural product present in the root extract of Lithospermum erythrorhizon, and the underlying mechanisms responsible for the inhibition of melanin synthesis in α-MSH-stimulated B16F10 cells and C57BL/6J mice was studied. Non-cytotoxic concentrations of (2-methylbutyryl)shikonin significantly repressed cellular tyrosinase activity and melanin synthesis in both in vitro and in vivo models (C57BL/6J mice). (2-Methylbutyryl)shikonin remarkably abolished the protein expression of MITF, tyrosinase, tyrosinase-related protein 1, and tyrosinase-related protein 2, thereby blocking the production of pigment melanin via modulating the phosphorylation status of MAPK proteins, viz., ERK1/2 and p38. In addition, specific inhibition of ERK1/2 attenuated the inhibitory effects of (2-methylbutyryl)shikonin on melanin synthesis, whereas selective inhibition of p38 augmented the inhibitory effect of BSHK on melanin synthesis. Moreover, topical application of (2-methylbutyryl)shikonin on C57BL/6J mouse tails remarkably induced tail depigmentation. In conclusion, with these findings, we, for the first time, report the hypopigmentation effect of (2-methylbutyryl)shikonin via inhibition of cellular tyrosinase enzyme activity, subsequently ameliorating the melanin production, thereby indicating that (2-methylbutyryl)shikonin is a potential natural therapy for hyperpigmentation disorders.

The in vitro and in vivo depigmentation activity of coenzyme Q0, a major quinone derivative from Antrodia camphorata, through autophagy induction in human melanocytes and keratinocytes.

BACKGROUND: Coenzyme Q0 (CoQ0), a novel quinone derivative of Antrodia camphorata, has been utilized as a therapeutic agent (including antioxidant, anti-inflammatory, antiangiogenic, antiatherosclerotic, and anticancer agents); however, its depigmenting efficiency has yet to be studied. METHODS: We resolved the depigmenting efficiency of CoQ0 through autophagy induction in melanoma (B16F10) and melanin-feeding keratinocyte (HaCaT) cells and in vivo Zebrafish model. Then, MPLC/HPLC analysis, MTT assay, Western blotting, immunofluorescence staining, LC3 transfection, melanin formation, GFP-LC3 puncta, AVO formation, tyrosinase activity, and TEM were used. RESULTS: CoQ0-induced autophagy in B16F10 cells was shown by enhanced LC3-II accumulation, ATG7 expression, autophagosome GFP-LC3 puncta, and AVOs formation, and ATG4B downregulation, and Beclin-1/Bcl-2 dysregulation. In α-MSH-stimulated B16F10 cells, CoQ0 induced antimelanogenesis by suppressing CREB-MITF pathway, tyrosinase expression/activity, and melanin formation via autophagy. TEM data disclosed that CoQ0 increased melanosome-engulfing autophagosomes and autolysosomes in α-MSH-stimulated B16F10 cells. CoQ0-inhibited melanogenesis in α-MSH-stimulated B16F10 cells was reversed by pretreatment with the autophagy inhibitor 3-MA or silencing of LC3. Additionally, CoQ0-induced autophagy in HaCaT cells was revealed by enhanced LC3-II accumulation, autophagosome GFP-LC3 puncta and AVO formation, ATG4B downregulation, ATG5/ATG7 expression, and Beclin-1/Bcl-2 dysregulation. In melanin-feeding HaCaT cells, CoQ0 induced melanin degradation by suppressing melanosome gp100 and melanin formation via autophagy. TEM confirmed that CoQ0 increased melanosome-engulfing autophagosomes and autolysosomes in melanin-feeding HaCaT cells. Treatment with 3-MA reversed CoQ0-mediated melanin degradation in melanin-feeding HaCaT cells. In vivo study showed that CoQ0 suppressed endogenous body pigmentation by antimelanogenesis and melanin degradation through autophagy induction in a zebrafish model. CONCLUSIONS: Our results showed that CoQ0 exerted antimelanogenesis and melanin degradation by inducing autophagy. CoQ0 could be used in skin-whitening formulations as a topical cosmetic application.

Alternative strategies for the recombinant synthesis, DOPA modification and analysis of mussel foot proteins - A case study for Mefp-3 from Mytilus edulis.

Mussel foot proteins (Mfps) possess unique binding properties to various surfaces due to the presence of L-3,4-dihydroxyphenylalanine (DOPA). Mytilus edulis foot protein-3 (Mefp-3) is one of several proteins in the byssal adhesive plaque. Its localization at the plaque-substrate interface approved that Mefp-3 plays a key role in adhesion. Therefore, the protein is suitable for the development of innovative bio-based binders. However, recombinant Mfp-3s are mainly purified from inclusion bodies under denaturing conditions. Here, we describe a robust and reproducible protocol for obtaining soluble and tag-free Mefp-3 using the SUMO-fusion technology. Additionally, a microbial tyrosinase from Verrucomicrobium spinosum was used for the in vitro hydroxylation of peptide-bound tyrosines in Mefp-3 for the first time. The highly hydroxylated Mefp-3, confirmed by MALDI-TOF-MS, exhibited excellent adhesive properties comparable to a commercial glue. These results demonstrate a concerted and simplified high yield production process for recombinant soluble and tag-free Mfp3-based proteins with on demand DOPA modification.

Synthesis and evaluation of 3-hydroxyquinolin-2(1H)-one derivatives as inhibitors of tyrosinase.

The tyrosinase (TYR) enzyme catalyses sequential reactions in the melanogenesis pathway: l-tyrosine is oxidised to yield L-3,4-dihydroxyphenylalanine (l-dopa), which in turn is converted to dopaquinone. These two reactions are the first two steps of melanin biosynthesis and are rate limiting. The accumulation or overproduction of melanin may cause skin hyperpigmentation and inhibitors of TYR are thus of interest to the cosmeceutical industry. Several TYR inhibitors are used to treat skin hyperpigmentation, however, some are ineffective and possess questionable safety profiles. This emphasises the need to develop novel TYR inhibitors with better safety and efficacy profiles. The small molecule, 3-hydroxycoumarin, has been reported to be a good potency TYR inhibitor (IC50 = 2.49 µM), and based on this, a series of eight structurally related 3-hydroxyquinolin-2(1H)-one derivatives were synthesised with the aim to discover novel TYR inhibitors. The results showed that four of the derivatives inhibited TYR from the champignon mushroom Agaricus bisporus (abTYR) with IC50 < 6.11 µM. The most potent inhibitor displayed an IC50 value of 2.52 µM. Under the same conditions, the reference inhibitors, thiamidol and kojic acid, inhibited abTYR with IC50 values of 0.130 and 26.4 µM, respectively. Based on the small molecular structures of the active 3-hydroxyquinolin-2(1H)-one inhibitors which are amenable to structure optimisation, it may be concluded that this class of compounds are good leads for the design of TYR inhibitors for cosmeceutical applications.

Design, synthesis, and anti-melanogenic efficacy of 2-mercaptobenzoxazoles with nanomolar tyrosinase activity inhibition.

Tyrosinase is a metalloenzyme that contains copper(II) ions. We designed and synthesized eight known low-molecular-weight 2-mercaptobenzoxazole (2-MBO) analogs as tyrosinase inhibitors. Our focus was on the mercapto functional group, which interacts with copper ions. Analogs 1-3 exhibited mushroom tyrosinase inhibitory activity at the nanomolar level and demonstrated strong potency with extremely low half-maximal inhibitory concentration (IC50) values of 80-90 nM for l-dopa and 100-240 nM for l-tyrosine. Analogs 2, 4, and 5 showed the most potent anti-melanogenic effects in B16F10 cells, and their mode of action was demonstrated by kinetic analysis. Their anti-melanogenic effects were similar to the tyrosinase inhibition results, suggesting that their anti-melanogenic effects could be attributed to their tyrosinase inhibitory ability. Experiments using copper-chelating activity assays and changes in tyrosinase inhibitory activity with and without CuSO4 demonstrated that 2-MBO analogs inhibit tyrosinase activity by chelating the copper ions of tyrosinase. In conclusion, the 2-MBO analogs show potential as anti-melanogenic agents with potent tyrosinase inhibitory activity.

Thiazol-4(5H)-one analogs as potent tyrosinase inhibitors: Synthesis, tyrosinase inhibition, antimelanogenic effect, antioxidant activity, and in silico docking simulation.

As the ß-phenyl-α,ß-unsaturated carbonyl (PUSC) structure was previously identified to play a key role in tyrosinase inhibition, 14 analogs with a PUSC structure built on a thiazol-4(5H)-one scaffold were synthesized using Knoevenagel condensation to serve as potential tyrosinase inhibitors. Through mushroom tyrosinase inhibition experiments, two analogs 9 and 11 were identified as potent tyrosinase inhibitors, with 11 exhibiting an IC50 value of 0.4 ± 0.01 µM, which indicates its 26-fold greater potency than kojic acid. Kinetic studies using Lineweaver-Burk plots revealed that 9 and 11 are competitive and mixed-type inhibitors, respectively; these kinetic results were supported by docking simulations. According to the B16F10 cell-based experiments, 9 and 11 inhibited melanogenesis more effectively than kojic acid due to their potent cellular tyrosinase inhibitory activity. In addition, analogs 9 and 11 exhibited moderate-to-strong antioxidant capacity, scavenging ABTS+, DPPH, and ROS radicals. In particular, analog 12 with a catechol moiety exhibited very strong ROS-scavenging activity, similar to Trolox. These results suggest that analogs 9 and 11, which exhibit potent tyrosinase inhibitory activity in mushroom and mammalian cells and anti-melanogenic effects in B16F10 cells, are promising antibrowning agents for crops and skin lightening agents for hyperpigmentation-related diseases.

Identifying genetic defects in oculocutaneous albinism patients of West Bengal, Eastern India.

BACKGROUND: Oculocutaneous albinism (OCA) is a congenital heterogeneous group of autosomal recessive disorders characterized by the absence or loss of melanin in the skin, eyes and hair of the affected individuals. Based on the mutated gene, OCA has been classified into eight sub-types (OCA1-8) with overlapping clinical phenotypes. Mutations in the TYR gene cause OCA1, the most prevalent OCA worldwide including India. Mutations in OCA2 and SLC45A2, both of which regulate melanosomal pH that is critical to TYR activity, cause OCA2 and OCA4 respectively, the other common OCA subtypes in India. METHODS: In the present study, we have included 54 OCA-affected cases from 41 unrelated families representing 16 different marriage/ethnic groups from 17 districts of West Bengal, India. We pursued a PCR-sequencing based approach followed by bioinformatic analysis to identify mutations in TYR, OCA2 and SLC45A2 genes. RESULTS: Mutations were detected in 27 of the 54 (50%) OCA patients from 18 unrelated families, representing 9 different marriage/ethnic groups from 11 districts of West Bengal. Three TYR variants: NM_000372.4: c.391 A > G, NP_000363.1: p. Lys131Glu; NM_000372.4: c.1037G > T; NP_000363.1: p. Gly346Val, NM_000372.4: c.715 C > T; NP_000363.1:p.Arg239Trp was identified for the first time in Eastern Indian OCA cases. A novel nonsense variant: NM_016180.5: c.389 T > A, NP_057264.4: p. Leu130* and a novel synonymous variation NM_016180.5: c.1092 A > G; NP_057264.4: p.364E = were identified in SLC45A2. Additionally, NM_016180.5: c.904A > T; NP_057264.4: p. Thre302Ser was identified for the first time in any Eastern Indian OCA case. We identified 2 previously reported mutations in OCA2. In concordance with previous reports, NM_000372.4: c.832C > T, NP_000363.1: p. (Arg278*) was the commonest TYR mutation. CONCLUSION: The results of our study enrich the mutational spectrum of the known OCA causing genes in Eastern India, which would facilitate accurate diagnosis, familial screening, carrier detection and containment of the disease load.

Viral Infection Induces Changes to the Metabolome, Immune Response and Development of a Generalist Insect Herbivore.

Host plant consumption and pathogen infection commonly influence insect traits related to development and immunity, which are ultimately reflected in the behavior and physiology of the insect. Herein, we explored changes in the metabolome of a generalist insect herbivore, Vanessa cardui (Lepidoptera: Nymphalidae), in response to both dietary variation and pathogen infection in order to gain insight into tritrophic interactions for insect metabolism and immunity. Caterpillars were reared on two different host plants, Plantago lanceolata (Plantaginaceae) and Taraxacum officinale (Asteraceae) and subjected to a viral infection by Junonia coenia densovirus (JcDV), along with assays to determine the insect immune response and development. Richness and diversity of plant and caterpillar metabolites were evaluated using a liquid chromatography-mass spectrometry approach and showed that viral infection induced changes to the chemical content of V. cardui hemolymph and frass dependent upon host plant consumption. Overall, the immune response as measured by phenoloxidase (PO) enzymatic activity was higher in individuals feeding on P. lanceolata compared with those feeding on T. officinale. Additionally, infection with JcDV caused suppression of PO activity, which was not host plant dependent. We conclude that viral infection combined with host plant consumption creates a unique chemical environment, particularly within the insect hemolymph. Whether and how these metabolites contribute to defense against viral infection is an open question in chemical ecology.

A turn-on fluorescence probe for imaging tyrosinase at the wound site in broken tail of zebrafish.

Tyrosinase (TYR) is a copper-containing oxidase that affects the synthesis of melanin in the human body, which is regulate to the pigmentation of the skin. Nevertheless, abnormal expression of TYR can lead to albinism, vitiligo and other skin diseases. Excessive accumulation of TYR is a marker of melanoma cancer and an important factor leading to pigmentation during wound healing, freckles and browning of fruits and vegetables. Efficient tracking of TYR is of significance for studying its pathophysiological mechanism. Herein, we synthesized a benzindole-based fluorescent probe Pro-OH to detect TYR in living cells and zebrafish. The probe displayed a high selectivity and sensitivity in distinguishing TYR from other analytes with the low detection limit of 1.024 U/mL. Importantly, Pro-OH was successfully used to imagine TYR at the wound site of broken tail of zebrafish.